We have succeeded in identifying a number of genes with antiretroviral activity, and the continuation of this project will naturally progress with a detailed examination of the mechanism of action of one validated restriction factor (Mx2) in Aim 1, and a more broadly based follow up of additional hits, as well as additional ISG screening in Aim 2. In Aim 1 we will build on our discovery that Mx2 is an antiretroviral protein by determining its mechanism of action. Mx2 inhibits HIV-1 infection in a capsid dependent manner at a step between reverse transcription and integration. A few potential mechanisms might underlie this activity. First, Mx2 might directly target the incoming viral capsid, in a manner akin to the TRIM5a and Fvl antiretroviral proteins Crucially, we have found a number of amino acids in CA that when mutated confer resistance to Mx2 antiviral activity. These will serve as key specificity controls in our binding and core-fate assays. Additionally, we will determine whether Mx2 binds to nuclear transport components, whether the nature of the Mx2 nuclear targeting signal is important for its antiviral activity and whether the route taken by HIV-1 into the nucleus affects its susceptibility to Mx2. Ultimately these experiments should determine how Mx2 functions and whether capsid recognition and/or specific nuclear entry pathway occlusion is responsible. Our ISG screening platform has revealed numerous (-30) candidate antiretroviral proteins that either protect cells from infection, or reduce the yield of infectious virons from ISG expressing cells. Currently, our analysis of these hits is at various stages of follow up. In Aim 2 we will follow the same paths exemplified by our previous work on Mx2 and CNP. Ultimately, we will aim to determine precisely how these ISGs inhibit reroviral replication, and crucially whether they are required for the full antiviral activity of interferon. Finally, we are screening additional ISGs that were not present in our originally constructed libraries. Again the path taken by these studies will mirror our previously successful approaches. RELEVANCE (See instructions): Mammals, including humans, have an array of antiviral defense mechanisms. The mechanism by which these antiviral defenses work is only beginning to be understood. Identifying and understanding the mechanism of action of antiretroviral gene products could lead to completely new chemotherapeutic strategies for tackling infectious diseases, including AIDS.